Carbohydrates exist in many forms in nature. In animals including man, examples include free reducing sugars in solution (such as the monosaccharide glucose in serum), free oligosaccharides in solution (such as the disaccharide lactose in milk), they can be attached to peptides or proteins through covalent linkages to a variety of amino acids (such as asparagine, serine, threonine and others), covalently attached to lipids such as ceramide (as in gangliosides) or attached to membrane anchors via phosphatidylinositols. Sugars are also found attached to many small molecules including some involved in metabolism, such as glucuronides. In the above examples, the length of the sugar chains can vary from one to over 100 sugar residues.
In lower organisms, including bacteria and plants, an even wider array of structures exists. The surface of bacterial cells can be covered by sugar polymers that are thousands of residues long, which can act as antigens in the detection of bacteria and as vaccines. Sugars are an integral part of bacterial cell walls. The sugars can themselves be antibiotics (such as the aminoglycoside antibiotics, for example streptomycin), or can be found as essential components of antibiotics (such as erythromycin and vancomycin), as enzyme inhibitors (as in Acarbose) or as anti-cancer agents (such as for example calicheamycin).
One area of particular interest is the structure of the carbohydrate chains (glycans) found attached to glycoproteins and glycolipids. The glycosylation pattern of glycoproteins has been shown to be important for their biological functions, including their bioavailablity, their targeting, and have even been directly correlated with the metastatic potential of tumor cells. The glycosylation pattern of human serum transferrin, for example, is being used as a diagnostic test for a series of genetic diseases termed Carbohydrate-Deficient Glycosylation Syndromes. Specific glycolipid sequences have been shown to be involved in neuronal development and cell surface signalling, in diabetes, and are accumulated in certain specific metabolic diseases such as Tay-Sachs, for which they are diagnostic.
The linkages between the sugar residues in the oligosaccharides and polysaccharides described above can have either the alpha or beta configurations, and the glycans can be multiply branched. The diversity of structures possible for glycan chains is therefore enormous and their structural characterization is therefore inherently complex. There is therefore a strong interest in methods for the detection, structural characterization, identification, quantitation, and chemical/enzymatic manipulation of carbohydrate and glycan structures, in research, in diagnostics, in monitoring the glycosylation of recombinant glycoproteins and in the development of new pharmaceutical agents. In this last context, the degree of terminal galactosylation and sialyation of the glycan chains of recombinant glycoprotein drugs such as erythreopoetin is critically important for its effectiveness.
Several methods are in current use for the analysis for carbohydrate structures, and these have recently been reviewed. Underivatized oligosaccharides and glycolipids can be analyzed by NMR-spectroscopy, by mass-spectrometry, and by chromatography. For the much larger glycoproteins, mass spectrometry provides more limited information but analysis of their proteolytic digests, i.e. glycopeptides, has been extensively used. Indirect structural information about underivatized oligosaccharides can also be deduced from their abilities to interact with carbohydrate-binding proteins such as lectins, antibodies or enzymes.
Carbohydrates themselves have no characteristic chromophores, only N-acetyl groups, so monitoring their separation by optical or spectroscopic detection is not commonly used. Pulsed amperometric detection of the polyols has however been an important technique for detection in chromatography. This technique has also been applied to the detection and identification of monosaccharides in solution.
The most widely used method for high-sensitivity detection of carbohydrates has been the labeling of the reducing ends (lactols, tautomers of hydroxyaldehydes and hydroxyketones) with either radioactive or fluorescent TAGs. Both chemical and enzymatic methods have been described that cleave carbohydrates from glycoproteins and glycolipids, permitting the generation of the required reducing sugars from glycoproteins (including monosaccharides released by exo-glycosidases or acid-hydrolysis), glycolipds and other glycoconjugates. Most commonly, such reducing sugars are reacted with amino-containing derivatives of fluorescent molecules under conditions of reductive amination: i.e., where the initially formed imines (C═N) are reduced to amines (CH—NH) to produce a stable linkage. In most cases, the labeling reactions have been performed in solution using a large excess of labeling agent. This requires separation of the excess labeling agent and its by-products prior to or during analysis. Other TAGs of utility in mass-spectrometry have been added in the same manner, by either amination or reductive amination, the detection then being performed by the mass-spectrometer.
Once the label has been added to permit specific detection, the carbohydrates (including monosaccharides) described above can subsequently be subjected to separation and detection/quantification. If specific glycosidases act on the tagged carbohydrates, they can cleave one or more sugar residues resulting in a change in chromatographic or electrophoretic mobility, as detected by, for example, a fluorescence detector in HPLC, CE or by a change in their mobility in SDS-PAGE, or a change in their mass as detected by a change in m/z value in a mass-spectrometer. Arrays of enzymes have been used to provide a higher throughput analysis.
Below a short overview of prior art is given:
Gao et al. 2003 reviews suitable techniques for derivatisation of carbohydrates in solution. In solution carbohydrates may be derivatised by reductive amination. In general, —NH2 groups of amines may react with aldehyde or ketone group of reducing sugars, thereby producing compounds of —C═N structure. Such compounds may further be reduced for example by NaCNBH3. Gao et al., 2003 does not disclose the capture or detection of terminal monosaccharides released from glycosylated substrates.
U.S. Pat. No. 5,100,778 describes a method for oligosaccharide sequencing comprising placing an identifying label on the reducing terminal residue of an oligosaccharide, dividing into a plurality of separate portions, treating each portion with for example specific glycosidases, pooling product and analysing the pools obtained. The document does not describe immobilised terminal monosaccharides.
U.S. Pat. No. 4,419,444 describes methods for chemically binding organic compounds containing carbohydrate residues to a support bearing reactive —NH2 groups. The methods involve either the periodate oxidation of carbohydrate diols to produce reactive aldehydes by cleaving of C—C bonds in the carbohydrate or oxidation of —CH2OH groups to —CHO groups enzymatically. Both oxidations will result in alteration of the structure of the carbohydrate. The reactive aldehydes can be immobilised by reaction with the —NH2 groups. After immobilisation of the carbohydrate containing compound a reduction step (for example using NaBH4) may be performed to increase stability. The document does not describe the immobilization of a single monosaccharide after cleavage from a carbohydrate-containing compound. Furthermore, the chemical nature of the carbohydrate has been altered and this alteration may impair further modulations, such as specific enzymatic cleavage by glycosidases. The document also does not describe the addition of any chemical reagents to the immobilised carbohydrates that result in the addition of molecular structures to it.
WO92/719974 describes a method of sequencing oligosaccharides. The method involves immobilising oligosaccharides on a solid support and subsequent treatment with a variety of glycosidases. Prior to immoblisation, the oligosaccharide may be linked to a conjugate. The document does not describe modulation of immobilised terminal monosaccharides, nor indeed treatment with the tagged compounds of the present invention.
Lohse et al. (“Solid-Phase Oligosaccharide Tagging (SPOT): Validation on Glycolipid-Derived Structures”, Angew. Chem. Int. Ed. 2006, 45, 4167-4172) disclose Solid-Phase Oligosaccharide Tagging on glycolipid-derived structures, however this article does not disclose cleavage of terminal monosaccharides from glycosylated substrates before analysis of the immobilised monosaccharides, nor indeed the use of the specific boronate compounds disclosed herein.
Various boronate compounds have been previously used for labelling and detecting carbohydrates in solution, such as disclosed in e.g. the following references:                “Boronic Acids: Preparation, Applications in Organic Synthesis and Medicine”, ed. Dennis G. Hall, pub. Wiley-VCH, in particular in chapters 12 and 13 (“Boronic Acid-based receptors and sensors for saccharides” and “Biological and medicinal applications of boronic acids”).        Yan et al., “Boronolectins and Fluorescent Boronolectins: An Examination of the Detailed Chemistry Issues Important for the Design”, Medicinal Reseach Reviews, Vol. 25, No. 5, 490-520, 2005        Mulla et al., “3-Methoxycarbonyl-5-nitrophenyl boronic acid: high affinity diol recognition at neutral pH”, Bioorganic & Medicinal Chemistry Letter 14 (2004) 25-27        Dowlut et al., “An Improved Class of Sugar-Binding Boronic Acids, Soluble and Capable of Complexing Glycosides in Neutral Water”, J. Am. Chem. Soc. 2006, 128, 4226-7        Hoeg-Jensen., “Preparation and Screening of Diboronate Arrays for Identification of Carbohydrate Binders”, QSAR Comb., Sci. 2004, 23        Boduroglu et al., “A colorimetric titration method for quantification of millimolar glucose in a pH 7.4 aqueous phosphate buffer”, Bioorganic & Medicinal Chemistry Letter 15 (2005) 3974-3977        Davis et al., “Simple and Rapid Visual Sensing of Saccharides”, Organic Letter, 1999 Vol. 1, No. 2, 331-334        He et al., “Chromophore Formation in Resorcinarene Solutions and the Visual detection of Mono- and Oligosaccharides”, J. Am. Chem. Soc. 2003, 124, 5000-5009        Gray et al., “Specific sensing between inositol epimers by a bis(boronate)”, Bioorganic & Medicinal Chemistry Letters 15 (2005) 5416-5418)        
However, none of the above-mentioned compounds have been used to label and detect carbohydrates immobilised to a solid support, nor indeed terminal monosaccharides attached to a solid support.
The above sections describe the biological importance and complexity of glycans, and summarizes some benefits of attaching TAGs such as boronates to sugars, including monosaccharides, although not to immobilised terminal monosaccharides after cleavage from a carbohydrate-containing molecule. To date, such TAG attachment has only been performed in solution using large excesses of tagging agent (and often additional chemical agents such as reducing agents), and thus require time consuming and frequently difficult separation techniques to be applied before either detection or further manipulation. There is therefore a great need for simple methods that can allow easier carbohydrate sequencing through identification of a terminal monosaccharide, without the need for complex methods for separating reaction starting materials, reagents, by-products and sought after products. We describe herein such simple methods.